4-trans-propenylveratrole

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 19 to June 08, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH (2011) EMA/CHMP/ICH/126642/2008. Guideline S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on February 05-07, 2014/ signed on April 24, 2014)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4-trans-propenylveratrole

EC Number:

228-958-7

EC Name:

4-trans-propenylveratrole

Cas Number:

6379-72-2

Molecular formula:

C11H14O2

IUPAC Name:

(E)-1,2-Dimethoxy-4-prop-1-en-1-ylbenzene

Test material form:

liquid

Details on test material:

- Physical state: Pale yellow, oily liquid
- Storage condition of test material: Room temperature

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 71 to 78 days
- Weight at study initiation: 331-393 g for males; 225-276 g for females
- Housing: Pre-pairing - 5/sex/cage; Pairing – 1 male:1 female per cage; Males after mating – 5 males/cage; Gestation – 1 female /cage; Lactation – 1 female/cage (+ litter); Solid (polycarbonate) bottom cages were used during the acclimatisation, gestation, littering and lactation periods; Grid bottomed cages were used during pairing.
- Diet: SDS VRF1 Certified powdered diet, ad libitum
- Water: Potable water taken from the public supply, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From January 19 to June 08, 2015

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Method of preparation: On each occasion of the preparation of the premix, the required amount of test substance was added to an equal amount of plain diet and stirred until visibly homogenous. This doubling up process was repeated until half of the final weight of premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 200 cycles to ensure the test substance was dispersed in the diet.
Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 200 cycles in a Turbula mixer.
- Frequency of preparation: Weekly
- Storage of formulation: Ambient temperature.

Duration of treatment / exposure:

Males were treated for a minimum of four consecutive weeks, including two weeks prior to pairing.
Females were treated for two weeks prior to pairing, throughout mating and gestation and until Day 7 of lactation.

Frequency of treatment:

Continuously

Post exposure period:

None

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

4 500 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

15 000 ppm (nominal)

No. of animals per sex per dose:

Samples of one femur were taken at necropsy from five male and female animals per group.

Control animals:

yes, plain diet

Positive control(s):

- Positive control: Cyclophosphamide
- Doses / concentrations: 20 mg/kg bw
- Route of administration: Oral

Examinations

Tissues and cell types examined:

Coded slides were examined by fluorescence microscopy and 4000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. At least one smear was examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear.
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
- The animals were killed by exposure to rising levels of carbon dioxide and one femur dissected out from each animal. The femurs were cleaned of all excess tissue and blood and the proximal epiphysis removed from each bone. The bone marrow of one femur from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration. The resulting cell suspensions were centrifuged at 1000 rpm (150 x g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976).

FIXATION AND SLIDE STAINING
1. Fixed for a minimum of 10 minutes in methanol and allowed to air-dry
2. Rinsed in purified water
3. Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
4. Washed in purified water for 5 minutes
5. Rinsed in cold tap water for 2 minutes
6. Stored at room temperature until required
7. Immediately prior to scoring, slides are wet mounted with coverslips using purified water

Evaluation criteria:

See section “Any other information on materials and methods incl. tables”

Statistics:

Each sex was analysed separately.
For the proportion of polychromatic erythrocytes, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.
For incidences of micronucleated polychromatic erythrocytes, an exact one-tailed Linear-by-Linear association test (Cytel 1995) with “step-down” was used on Groups 1 to 4 for an increase from control. If significant, then the analysis was carried out on Groups 1 to 3. Exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.
If the exact version of a test could not be calculated (due to the amount of data), then the asymptotic version was used instead.
Statistical significance was declared at the 5% level for all tests.
The data were received in an Excel document and analysed using SAS 9.1.3 (SAS Institute Inc., 2002) (Jonckheere's and Wilcoxon tests) and StatXact 3 (Cytel 1995) (Linear-by-Linear and Permutation tests).

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Test item did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes (MPCE) or micronucleated normochromatic erythrocytes (MNCE) in male or female rats.
- Proportion of polychromatic erythrocytes (%PCE): Test item did not cause any statistically significant decreases in the proportion of polychromatic erythrocytes in male or female rats.

- Positive control: The coded positive control slides prepared from study HLS1035 demonstrated the ability of the scorer to detect increases in micronucleated polychromatic erythrocytes.

Any other information on results incl. tables

Table 7.6.2/1: Summary of results and statistical analysis

Treatment	Dosage	Proportion of PCE % # (SD)	Incidence MPCE mean # (SD)	Group mean % MPCE #
Male data
Vehicle	0	49.5 (3.5)	5.6 (1.7)	0.14
Test item	1500 ppm	49.6 (2.9)	7.0 (2.9)	0.18
	4500 ppm	49.6 (2.3)	4.8 (3.1)	0.12
	15000 ppm	48.9 (2.7)	4.8 (3.0)	0.12
Cyclophosphamide^a	20 mg/kg	44.3* (3.1)	80.8** (11.6)	2.02
Female data
Vehicle	0	61.2 (4.9)	8.8 (2.7)	0.22
Test item	1500 ppm	60.3 (6.8)	9.2 (1.8)	0.23
	4500 ppm	68.7 (3.7)	6.2 (3.7)	0.16
	15000 ppm	63.0 (5.6)	7.0 (1.4)	0.18
Cyclophosphamide^a	20 mg/kg	44.1** (2.6)	64.4** (12.6)	1.61

 

Vehicle: Basal diet

PCE: Polychromatic erythrocytes

MPCE: Number of micronucleated polychromatic erythrocytes observed per 4000 polychromatic erythrocytes examined

SD: Standard deviation

^a Positive control slides from HLS1035

# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table.

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

*p< 0.05 (significant)

**p< 0.01 (significant)

otherwise p> 0.05 (not significant)

Applicant's summary and conclusion

Conclusions:

Under the test conditions, test item did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female Crl:CD(SD) rats.

Executive summary:

In micronucleus test conducted according to OECD 474 guideline and in compliance with GLP, induction of micronuclei by test item in bone marrow cells of male and female Crl:CD(SD) rats was assessed.

 

Bone marrow smears were obtained from 5 male and 5 female animals in the vehicle control group and in each of the test substance groups at the appropriate time. At least one smear from each animal was examined for the presence of micronuclei in 4000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

 

No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes and no statistically significant decreases in the proportion of polychromatic erythrocytes were observed in Crl:CD(SD) rats at any dose level, compared to vehicle control values. The coded positive control slides prepared from study HLS1035 demonstrated the ability of the scorer to detect increases in micronucleated polychromatic erythrocytes.

 

Under the test conditions, test item did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female rats.